Cinzia Ricci

p185neu protein is required for tumor and anchorage-independent growth, not for cell proliferation of transgenic mammary carcinoma

Transgenic FVB‐NeuN mice (N202) bearing the rat neu protooncogene driven by the mouse mammary tumor virus promoter/enhancer develop focal mammary carcinomas overexpressing the neu‐encoded p185neu protein. In vitro expression of p185neu among mammary carcinoma cultures was heterogeneous, and we could establish some cell lines and clones displaying a complete loss of p185neu expression, along with others with very high p185neu protein level. Upon in vivo injection, p185neu‐positive cells gave rise to fast‐growing tumors with a short latency, while p185neu‐negative cells required a very long latency time, and the resulting tumors were invariably p185neu‐positive. The lower growth ability of p185neu‐negative cells in vivo was also confirmed in athymic nude mice. In vitro, analysis of anchorage‐independent growth in soft agar revealed colony formation from p185neu‐positive but not p185neu‐negative cells. The direct involvement of p185neu in clonogenicity was demonstrated by the inhibition of p185neu‐positive colony growth in soft agar in the presence of an anti‐p185neu monoclonal antibody. By contrast, a higher level of anchorage‐dependent clonogenic growth and proliferation was observed in p185neu‐negative cells as compared to p185neu‐positive cells, thus explaining the relative ease with which p185neu‐negative cell lines and clones were established in vitro. Together, the results indicate that p185neu expression can lead to tumor formation and metastasis through the modification of intrinsic properties of cells related to anchorage‐independent growth ability rather than to proliferation or host‐dependent mechanisms. Int. J. Cancer 87:186–194, 2000.

The Metastatic Ability of Ewing’s Sarcoma Cells Is Modulated by Stem Cell Factor and by Its Receptor c-kit

Ewings sarcoma is a primitive highly malignant tumor of bone and soft tissues usually metastasizing to bone, bone marrow, and lung. Growth factor receptors and their ligands may be involved in its growth and dissemination. We analyzed the expression of c-kit and its ligand stem cell factor (SCF) in a panel of six Ewings sarcoma cell lines. All cell lines exhibited substantial levels of surface c-kit expression, and five of six displayed transmembrane SCF on the cell surface. Expression of c-kit was down-modulated in all lines by exposure to exogenous SCF. The SCF treatment was able to confer to cells a growth advantage in vitro, due both to an increase in cell proliferation and to a reduction in the apoptotic rate. When used in the lower compartment of a migration chamber, SCF acted as a strong chemoattractant for Ewings sarcoma cells. The pretreatment of cells with SCF reduced their chemotactic response to SCF. In athymic nude mice, Ewings sarcoma cells injected intravenously metastasized to the lung and to a variety of extrapulmonary sites, including bone and bone marrow. Metastatic sites resembled those observed in Ewings sarcoma patients and corresponded to SCF-rich microenvironments. The in vitro pretreatment of cells with SCF strongly reduced the metastatic ability of Ewings sarcoma cells, both to the lung and to extrapulmonary sites. This could be dependent on the down-modulation of c-kit expression observed in SCF-pretreated cells, leading to a reduced sensitivity to the chemotactic and proliferative actions of SCF. Our results indicate that the response to SCF mediated by c-kit may be involved in growth, migration, and metastatic ability of Ewings sarcoma cells.

Identification of new genes related to the myogenic differentiation arrest of human rhabdomyosarcoma cells

Rhabdomyosarcoma is a soft tissue tumor committed to the myogenic lineage but arrested prior to terminal differentiation. To identify new genes implicated in the block in myogenic differentiation of rhabdomyosarcoma cells and those responsible for their proceeding along the myogenic pathway we used cDNA microarrays to compare gene expression profiles of two clones of the human embryonal rhabdomyosarcoma cell line RD with different myogenic potentials: RD/12, which is unable to differentiate, and RD/18, which shows elements able to terminally differentiate, as defined by the expression of myosin heavy chain (up to 50% of the population) and the formation of multinucleated myotube-like structures. We identified 80 genes differentially expressed by the two clones. Differentiating RD/18 cells overexpressed the myogenic transcription factor myogenin along with known myogenic markers; myogenin transfection into undifferentiated RD/12 cells was able to revert the phenotype giving rise to 94% of clones displaying a differentiated morphology. RD/18 cells also expressed several genes not known to be expressed in rhabdomyosarcoma or muscle cells, such as pigment-epithelium derived factor and endothelin-3. Poorly differentiated RD/12 cells, along with genes related to mesenchymal lineage or early myogenic commitment, also expressed genes not previously known to be related to the differentiation block of human rhabdomyosarcoma, such as monocyte chemotactic protein-1, connective tissue growth factor and insulin-like growth factor binding protein-5. Differential expression of these genes in a time course of differentiation suggested their potential roles as either new myogenic markers or repressors of differentiation. These results identify a cluster of new genes related to the aberrant myogenic differentiation program of human rhabdomyosarcoma cells.

Expression of HER/erbb family of receptor tyrosine kinases and induction of differentiation by glial growth factor 2 in human rhabdomyosarcoma cells

Five human rhabdomyosarcoma cell lines were used to investigate the surface expression of HER/erbB receptors, particularly of HER‐2, HER‐3 and HER‐4, by flow cytometry. HER‐2 was expressed in 3/5 rhabdomyosarcoma cell lines. HER‐3 was expressed in all rhabdomyosarcoma cell lines. None of the rhabdomyosarcoma cell lines investigated showed HER‐4 surface expression. To study the biological activity of HER/erbB receptors in rhabdomyosarcomas, cells were cultured in the presence of glial growth factor 2 (GGF‐2), a specific ligand of HER‐3 that is able to stimulate myogenesis in normal myoblasts. In 3 of 3 human rhabdomyosarcoma cell lines positive for both HER‐2 and HER‐3 receptors, the treatment with GGF‐2 induced a significant increase in myogenic differentiation. Int. J. Cancer 87:29–36, 2000.

Photocontact dermatitis from UV filters

UV filters are currently the most common photoallergens (1, 2), particularly the benzophenones (2–7). Recently, the concentration of UV filters in the GIRDCA photopatch test series has been increased from 2% to 10% pet.

Prevention of HER‐2/neu transgenic mammary carcinoma by tamoxifen plus interleukin 12

FVB‐NeuN (N#202) female mice transgenic for the HER‐2/neu protooncogene driven by the murine mammary tumor virus (MMTV) promoter develop mammary carcinomas with a progression from focal atypical hyperplasia to in situ carcinoma and to invasive carcinoma that closely resembles that of human neoplasia. Here we report that the combination of tamoxifen plus interleukin 12 (IL‐12) results in a very effective prevention of mammary carcinogenesis, significantly higher than those obtained with either tamoxifen or IL‐12 alone. At 1 year of age, 20% of control mice resulted tumor‐free, whereas 80% of mice receiving the combined treatment were tumor‐free. At 2 years of age, less than 5% of control mice were tumor‐free, as opposed to 70% of mice treated with tamoxifen plus IL‐12. The combined treatment inhibited mammary carcinogenesis mainly through a reduction in the number of mammary cells at risk of progression, a reduction in estrogen receptors (ERs) expression and a reduction in the angiogenic support to mammary development, likely due to cross‐talk between tamoxifen and interferon‐γ (IFN‐γ) (the main downstream mediator elicited in vivo by IL‐12). The addition of IL‐12 to the tamoxifen treatment more than doubled mouse lifetime and did not exacerbate known side effects of tamoxifen.

HER/erbB receptors as therapeutic targets of immunotoxins in human rhabdomyosarcoma cells.

Human rhabdomyosarcoma cells express HER/erbB growth factors receptors. Receptors belonging to this family are overexpressed and play a role in many types of epithelial and neural cancer and have been selected as targets for cancer therapy. In this paper EGF-R, HER-2 and HER-3 receptors were tested as therapeutic targets of immunotoxins in human rhabdomyosarcoma. Rhabdomyosarcoma cells were treated with indirect immunotoxins consisting in primary specific murine monoclonal antibodies recognizing EGF-R, HER-2 and HER-3 followed by secondary F(ab´)2 antimouse immunoglobulin linked to saporin-S6, a type 1 ribosome-inactivating protein (RIP) from the seeds of Saponaria officinalis. The indirect immunotoxin targeting EGF-R caused a significant inhibition in cell growth and protein synthesis and a strong increase in apoptosis in rhabdomyosarcoma cells, whereas indirect immunotoxins against HER-2 and HER-3 were ineffective. The toxic activity of anti-EGF-R immunotoxin was also observed on rhabdomyosarcoma cells expressing low level of EGF-R. EGF-R could be a novel therapeutic target of immunotoxins in human rhabdomyosarcoma.

An anti-apoptotic role for NGF receptors in human rhabdomyosarcoma

The expression and biological function of Nerve Growth Factor (NGF) receptors was studied in a panel of rhabdomyosarcoma cell lines derived from embryonal and alveolar histotype. All the cell lines expressed both the high affinity receptor TrkA and the low affinity receptor p75(NTR). Treatment with exogenous NGF did not considerably alter rhabdomyosarcoma cell growth or differentiation, but significantly inhibited spontaneous apoptosis as well as apoptosis, and induced by serum starvation or apoptosis induced by treatment with cycloheximide (CHX). Rhabdomyosarcoma cell lines expressed NGF and other neurotrophins and trace amounts of NGF protein were found in the supernatants of rhabdomyosarcoma cell cultures. Blocking the putative autocrine loop with an anti-NGF antibody resulted in an increase in apoptosis compared with control cultures. These data suggest that the simultaneous presence of both high and low affinity NGF receptors engaged by endogenous or exogenous NGF might contribute to the escape from apoptosis exhibited by the rhabdomyosarcoma cells.

Contact sensitization to sunscreens

BACKGROUND Para aminobenzoic acid (PABA) derivatives and cinnamate are chemical sunscreens that protect against UVB (290 to 320 nm). They may occasionally produce contact and photocontact sensitization. OBJECTIVE To report a sensitization to octyl-dimethyl-PABA and photosensitization to 2-ethylhexyl-p-metossicinnamate in a 31-year-old man. METHODS A patient with a 3-year history of a relapsing dermatitis involving the face, neck, legs, and knees is reported. The eruption had recurred every summer after sunlight exposure. Patch tests with International Contact Dermatitis Research Group (IC-DRG) standard series and the photopatch series (Hermal-Trolab, Reinbek, Germany) using Finn chambers on Scanpor (Norgesplaster A/S, Oslo, Norway) were carried out. RESULTS We found a positive reaction to Balsam of Peru, fragrance mix, Escalol 507, and Parsol MCX (Hermal-Trolab, Reinbek, Germany). Photopatch test revealed a positive reaction only for Parsol MCX. CONCLUSION The incidence of allergic contact dermatitis to sunscreens is considered low. Recently sunscreens patch test concentrations have been increased from 2% to 10%. These higher percentages will probably permit the identification of more cases of sunscreens allergy in the near future.

Facial contact dermatitis due to metronidazole

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